TGF-β inhibitor treatment of H₂O₂-induced cystitis models provides biochemical mechanism for elucidating interstitial cystitis/painful bladder syndrome patients

Interstitial cystitis/painful bladder syndrome (IC/PBS) is a chronic disease for which no effective treatment is available. Transforming growth factor-β (TGF-β) is thought to be involved in the pathogenesis of IC/PBS, and previous studies have suggested that administrations of a TGF-β inhibitor significantly ameliorated IC/PBS in a mouse model. However, the molecular mechanisms underlying the therapeutic effect of a TGF-b inhibitor on IC/PBS has not been comprehensively analyzed. TGF-β has a variety of actions, such as regulation of immune cells and fibrosis. In our study, we induced IC/PBS-like disease in mice by an intravesical administration of hydrogen peroxide (H₂O₂) and examined the effects of three TGF-β inhibitors, Repsox, SB431542, and SB505124, on the urinary functions as well as histological and gene expression profiles in the bladder. TGF-β inhibitor treatment improved urinary function and histological changes in the IC/PBS mouse model, and SB431542 was most effective among the TGF-β inhibitors. In our present study, TGF-β inhibitor treatment improved abnormal enhancement of nociceptive mechanisms, immunity and inflammation, fibrosis, and dysfunction of bladder urothelium. These results show that multiple mechanisms are involved in the improvement of urinary function by TGF-β inhibitor.


Introduction
Interstitial cystitis/painful bladder syndrome (IC/PBS) is a chronic disease characterized by bladder pain, frequent and urgent urination, and nocturia.The known prevalence of IC/PBS or conditions suggestive of IC/PBS ranged from 0.01% to 2.3%, of the population, with an approximately five-fold dominance among the women.IC/PBS is an intractable illness for which there is currently no effective treatment [1].
IC/PBS is considered to be caused by various pathological mechanisms including dysfunction of urothelium, activation of lymphocytes and mast cells, bladder inflammation, abnormal enhancement of nociceptive mechanisms, toxic urine substances, microbial infection, etc. IC/ PBS is divided into three groups: Hunner type interstitial cystitis (HIC), Non-Hunner type interstitial cystitis (NHIC), and hypersensitive bladder (HSB).HIC has been reported to be accompanied by significant lymphocytic infiltration and urothelial cell denudation in the bladder, which are not seen in NHIC and HSB [1].
A commonly used animal model for IC/PBS is cyclophosphamide (CP)-induced cystitis in mice [2].An intraperitoneal injection of CP induces acute hemorrhagic cystitis with bladder overactivity.However, this cystitis model is characterized by acute inflammation rather than chronic pathology.In the CP-induced cystitis, inflammation sustains for only short periods and ceases within several days.Recently, a mouse model using H 2 O 2 has been reported.Even a week after an infusion of H 2 O 2 into the bladder, urination frequency remains high, and the high frequency of urination lasts for longer periods than that in CP model [3].
Transforming growth factor-β (TGF-β) has been reported to increase in the bladder tissue of IC/PBS patients [4].Some previously studies showed that an intravesical administration of TGF-β receptor inhibitor (SB505124) 48 hours after induction of cystitis decreased the frequency of urination and increased one-time voiding volume in mice.They also reported that TGF-β inhibitors reduced ATP release from urothelial cells and inhibited bladder afferent nerve discharge via stimulation of purine receptors [5,6].However, TGF-β signaling activates multiple and complicated intracellular pathways and affect the expression regulation of a variety of genes [7,8].The overall mechanism is not clear as to how the inhibition of TGF-β signaling ameliorates the cystitis.In addition, long-term effect of TGF-β inhibitors remains to be clarified.Therefore, we administered various TGF-β inhibitors to H 2 O 2 -induced cystitis mice, and analyzed urinary disturbance for 7 days.We also performed comprehensive transcriptome analyses to elucidate the overall mechanisms of the treatment of H 2 O 2 -induced cystitis by TGF-β inhibitors.

Chemical compounds
SB431542 and SB505124 were purchased from Cayman chemical company (Ann Arbor, MI, USA).Repsox was purchased from focus biomolecules (Plymouth Meeting, PA, USA).

Cystitis mouse model and treatment with TGF-β inhibitor
All animal experiments were approved by the Committee for Animal Research, Kyoto Prefectural University of Medicine (M2021-329), and the care of the animals was performed according to the institutional guideline and Guide for the Care and Use of Laboratory Animals.Female C57BL/6N mice were purchased from SHIMIZU Laboratory Supplies (Kyoto, Japan).They were housed in groups of 4 per cage in a room maintained at 23˚C and 50% relative humidity with an alternating 12-h light/dark cycle (the lights came on automatically at 7:00 a. m.).Food and water were freely given.The H 2 O 2 -induced cystitis model was prepared as previously reported, with slight modification [3].Briefly, 30% H 2 O 2 (Nacarai tesque) was diluted with sterile saline to 1.5% immediately before use.The mice at the age of 8 weeks were anesthetized with isoflurane.A 24-gauge catheter was inserted into the bladder through the urethra and 100μL of 1.5% H 2 O 2 solution was infused into the bladder for 30 minutes.Urinary frequency and one-time voiding volume were measured on days 1 and 8 to evaluate the effect of the H 2 O 2 irritation on urinary function.Thirty min after H 2 O 2 emission, 1.2 mM TGF-β inhibitors (either SB431542, SB505124, or Repsox) were intraperitoneally injected at 100μl/ mouse.TGFβ inhibitor concentrations were determined based on previous literature [9][10][11].In this experiment of TGF-β inhibitor treatment, 100μL of saline was infused into the bladder of the control mice.On day 4, an injection of TGF-β inhibitors were repeated.

Mouse voiding behavior analysis
Urination was recorded by the Voided Stain On Paper (VSOP) method as described with slight modifications(S1 Fig) [12].To avoid micturition variation resulting from circadian rhythms, functional tests were conducted at similar times of the day (between 9:00 AM and 1:00 PM).Briefly, each mouse was placed in a certain area above a filter paper (Advantec 300 mm; Tokyo Roshi Kaisha, Ltd., Tokyo, Japan).The voiding time and area were recorded for 3 hours.The recorded urine stains were scanned into image files and the stained areas were calculated using the software ImageJ v1.47 (National Institutes of Health, Bethesda, MA, USA).

Hematoxylin and eosin (HE) staining
Mice were sacrificed by intraperitoneal administration of 100-150 mg/kg of pentobarbital and bladder was excised from the mice 3 hours, 1 day and 8 days after H 2 O 2 infusion.Bladder tissue was dissected in an axial direction.The tissue was fixed in 4% paraformaldehyde (PFA).After fixation in 4% PFA some parts of the organ were cryoprotected in 30% sucrose PBS (-) and freeze-mounted.The specimens were sliced into 10μm thick sections and stained with hematoxylin and eosin.

RNA-seq analyses
Total RNA was extracted from a hemisphere of the bladder using the ISOGEN II (NIPPON GENE CO., LTD) according to the manufacturer's instructions.mRNA was purified from total RNA using poly-T oligo-attached magnetic beads, and after random fragmentation, cDNA was synthesized.The qualified libraries were sequenced with Illumina Novaseq 6000 (Illumina; paired-end, 150 bp).Analysis was performed using strand NGS (https://www.strand-ngs.com/).

Azan staining
Bladder was excised from the mice 10 days after H 2 O 2 administration.Bladder was cut at the maximum plane in the axial direction and tissue sections were subjected to Azan staining.
The specimens were sliced into 10μm thick sections.Firstly, the tissues were mordanted for 30 min in the mordant solution which consists of 10% potassium dichromate and 10% trichloroacetic acid.Next, they were washed with water, the tissues were incubated in amocarzine G solution at room temperature for 30 min, washed with water, immersed in 1% aniline ethanol, washed with acetate alcohol, washed with water, and they were stained in 5% phosphotungstic acid overnight.Next day, they were washed with water and stained in aniline blue-orange G solution for 1 hour.Subsequently, they were dehydrated in absolute ethanol, transparentized in Xylene, mounted with neutral resin.The percentage of bladder tissue affected by fibrosis was determined by calculating the ratio of collagen area to smooth muscle area.Quantitative analysis of the collagen area was performed using an image analyzer system (Fiji 2.5.0;National Institutes of Health, Bethesda, MA, USA).

Statistical analyses
Data are expressed as means +/− SD.Statistical significance was analyzed by one-way ANOVA with the Tukey-Kramer post hoc test.P < 0.05 was considered significant.All analyses were conducted with EZR.volumes of all the groups, suggesting that the increase in urinary frequency of the TGF-β-treated mice was not due to high total urine volume.These results strongly suggest SB431542 as the most effective TGF-β inhibitor for the treatment of impaired urinary functions in the cystitis mice.

Possible involvement of inflammatory and neural signaling-related genes in the therapeutic effect of SB431542 on mouse cystitis
To find out the molecular mechanisms through which a TGF-β inhibitor alleviated the cystitis, we performed RNA sequencing (RNA-seq) analysis for the bladder tissue of control (sham),  The correlation coefficient between the control and the H 2 O 2 /SB431542-treated groups was the highest (R2 = 0.932), followed by that between the H 2 O 2 /SB431542-treated and the H 2 O 2 /saline-treated groups (R2 = 0.878).The comparison between the control and the H 2 O 2 / saline-treated groups represented the lowest correlation coefficient value (R2 = 0.765).Thus, it was considered that the irritable effects of H 2 O 2 were counteracted by the SB431542 administration through the normalization of the aberrant gene expression.Among the Gene Ontology (GO) terms categorized into "biological process", we listed ten GO terms that represent gene sets differentially expressed between two groups (Fig 4C).Nine of the ten GO terms for the gene sets that showed highest p values for control group vs. H 2 O 2 /saline-treated were related to "immunity and inflammation", suggesting that the effects of H 2 O 2 were heavily associated with "immunity and inflammation".In addition, four of the ten GO terms for the gene sets showing highest p values for H 2 O 2 /saline vs. H 2 O 2 /SB431542-treated groups were related to in "synaptic signal", which may imply that synaptic signals may play important roles in the effects of SB431542 on the cystitis.There was also a significant difference in expression levels of the genes involved in the "immunity and inflammation" between the control and H 2 O 2 / SB431542-treated groups, but the p-value was not as low as that for the difference for the control vs. H 2 O 2 /saline-treated groups.Based on the results of GO analyses, we performed heatmap and hierarchical clustering analyses for genes involved in "bladder afferents nerve" and those involved in "immunity and inflammation", as well as for genes representing "keratin" and "collagen" that may be involved in the pathogenesis of IC/BPS and the effect of TGF-β inhibitors (Fig 4D).The heatmap and hierarchical clustering analysis revealed that the H 2 O 2 / SB431542-treated group is more closely similar to the control group than the H 2 O 2 /salinetreated group with respect to the genes related to "bladder afferents nerve" and "immunity and inflammation".The gene sets related to "keratin" and "collagen" were highly expressed in the H 2 O 2 /saline-treated group, but their expression was decreased by SB431542 administration, as demonstrated by the similarity between the heat maps for the control and H 2 O 2 / SB432542-treated groups.

H 2 O 2 infusion augmented expression of Trpa1, IL-1β, Col7a1, Krt13, and Krt6a genes in the bladder, which was reversed by SB431542 administration
To confirm the results from the heatmap and the hierarchical clustering analyses, we performed reverse transcription-quantitative PCR (RT-qPCR) and validated expression levels of the representative genes that were categorized in the "bladder afferents nerve", "immunity and inflammation", "keratin", and "collagen".We found that mRNA for the Trpa1 gene that is involved in bladder afferents was highly expressed in the bladder of the H 2 O 2 /saline-treated mice, whereas the Trpa1 mRNA was significantly less abundant in the bladder of control and the H 2 O 2 /SB431542-treated mice.Similar results were also obtained by evaluating mRNA for the inflammatory cytokine IL-1β, Krt13 and Krt6a that are expressed in urothelial cells, and Col7a1 that is involved in fibrosis (Fig 5).

SB431542 administration ameliorated bladder fibrosis in the H 2 O 2induced cystitis model
To determine the degree of fibrosis, the bladder tissue sections were subjected to AZAN staining that enables us to distinguish between collagen fibers (blue) and muscles (red).The proportion of the fibrosis area in each bladder tissue specimen was calculated (Fig 6).The H 2 O 2 / saline-treated group higher proportion of fibrosis area in the bladder than the control group, while the proportion in the H 2 O 2 /SB431542-treated group was comparable to that in the control group.

Discussion
Systemic administration of a TGF-β inhibitor improved disturbance of urinary function in a mouse model of IC/PBS.The present results can contribute to the elucidation of molecular pathophysiology of IC/PBS and mechanisms of therapeutic effect of TGF-β inhibitors on IC/ PBS [7].
Our RNA-seq analyses results suggest that some genes categorized into four gene sets, i.e., "bladder afferents nerve", "immunity and inflammation", "keratin", and "collagen" may have contributed to the therapeutic effect of SB431542 on the cystitis.
One of the mechanisms of bladder pain induction in IC/PBS is assumed to be chronic tissue inflammation that causes functional changes in C-fiber afferents [13].In particular, Trpa1, a polymodal receptor, has been identified not only in C-fiber afferents, but also in urothelial cells and detrusor muscle cells in the urinary bladder.It has been confirmed that an administration of a TRPA1 channel activator into the bladder induces detrusor overactivity [14].
The H 2 O 2 stimulus causes oxidative stress in the bladder, leading to activation of the afferent pathway of capsaicin-sensitive C-fibers, which subsequently results in hyperactivity of the detrusor muscles [15,16].Oyama et al. reported that mRNA expression of Trpa1 in bladders of the H 2 O 2 -treated mice was significantly higher than that in the control mice [17].In the present study, the overexpression of the genes that could be involved in afferent nerve of the bladder was restored by SB431542 treatment, as shown by the heatmap of RNA-seq analyses (Fig 4D).RT-qPCR indicated that the mRNA level for Trpa1 was elevated in the cystitis and was decreased by SB431542 treatment (Fig 5).
Recent RNA-seq analyses suggested that immunologic inflammatory processes might support the pathophysiology of Hunner type interstitial cystitis [18].TGF-β has opposing proand anti-inflammatory effects.TGF-β knockout mice displayed lethal inflammation, whereas an immunosuppressive function of TGF-β is also reported [19,20].Meanwhile TGF-β may conversely play pro-inflammatory effects on Th17 cells and cells of the innate immune system.TGF-β, together with interleukin-6 (IL-6), was reported to be an essential player in driving pro-inflammatory Th17 lineage differentiation [21][22][23].In addition, patients with autoimmune diseases often develop IC/BPS as a complication [24].Th17 cells, which is causally associated with Hunner type interstitial cystitis, have also been implicated in autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, psoriasis, and inflammatory bowel disease [25][26][27].A previous report suggested that inhibition of the TGF-β signaling pathway may be beneficial in patients with autoimmune disorders, such as multiple sclerosis, through downregulation of the Th17 pathway [28].Minami et al. reported that mRNA expression of IL-1β in bladders of the H 2 O 2 -treated mice was significantly higher than that in the control mice [29].
In the present study, the heatmap and hierarchical clustering analysis showed that TGF-β inhibitor treatment partially restored the expression of the genes that are involved in immunity and inflammation, as shown in the Fig 4D .Also, the mRNA level for IL-1β was elevated in the cystitis and was decreased by SB431542 treatment (Fig 5).However, GO analysis indicated that there was still an inflammatory difference between control and H 2 O 2 /SB431542-treated group.In other words, it can be concluded that the administration of TGF-β inhibitors partially, but not completely, suppressed inflammation.
TGF-β inhibitors have been reported to inhibit fibrosis in various diseases [7].There was a report that a TGF-β receptor inhibitor was administered to rats with ketamine-induced bladder fibrosis and it inhibited the bladder fibrosis of the mouse [30].Minami et al. showed that fibrosis area in bladder submucosa of the H 2 O 2 -treated mice increased than that of the control mice [29].In IC/BPS patients, collagen staining area of the bladder is increased.The bladder fibrosis is associated with decreased bladder compliance, uncontrolled detrusor contractions, increased frequency of urination, and decreased bladder capacity [31][32][33][34].In the present study, TGF-β inhibitor administration improved the expression of genes related to fibrosis as shown in the heatmap of RNA-seq analyses.Moreover, SB431542 treatment inhibited fibrosis of the bladder in cystitis (Fig 6).RT-qPCR indicated that the mRNA level for Col7a1, of which mutation is known to cause hereditary epidermolysis bullosa, was elevated in the cystitis and was decreased by SB431542 treatment [35].Although Col7a1 has not been reported as a mediator of H 2 O 2 -induced cystitis, the results of increased Col7a1 expression in H 2 O 2 -induced cystitis mice are consistent because the bladder wall of H 2 O 2 -induced cystitis mice has been shown to have increased fibrosis [29].
Urothelial denudation is another characteristic histological feature of Hunner type interstitial cystitis.The loss of the urothelial barrier could permit the stimulation of urine to directly contact the afferent nerves in the bladder [18].Since a defect in the urothelium is thought to be one of the causes in IC/PBS, there is a report of transplantation of urothelial cells created by direct conversion into H 2 O 2 models [36].Keratin is one of the cytoskeletons expressed in epithelial cells, including the urothelium of the bladder.Laguna et al. reported that keratin profiles of IC/BPS and normal bladder are different from each other [37].Krt13 is a keratin that can be expressed in normal urothelium and in IC/BPS to the same extent, but Krt6a, a component of basal keratin, is expressed in urothelium in IC/PBS but not in normal urothelium [37][38][39].In the present study, mRNA levels for Krt13 and Krt6a were increased by H 2 O 2 administration and decreased by TGF-β inhibitor administration, suggesting that TGF-β inhibitor may have protected urothelial cell damage by H 2 O 2 stimulus.Krt6 and Krt13 has not been reported as a mediator of H 2 O 2 -induced cystitis.The Krt6 expression is seen in stratified epithelial cells featuring hyperproliferation, such as psoriasis.Krt13 is associated with epithelial cell differentiation and signal transduction [40].H 2 O 2 destroys urothelial cells, followed by regeneration of urothelial cells through a process of proliferation and differentiation.Therefore, it seems that these increases in mRNA expression of keratins in H 2 O 2 -induced cystitis mice are consistent.According to previous reports, proteins involved in urothelial barrier function include uroplakins, proteoglycan core proteins, tight junction proteins, and claudins [1,41].Uroplakin knockout mice have been used as mouse models of IC/PBS [41].S4 Fig shows that the mean raw reads count of H 2 O 2 -treated mice was lower than that of control mice and SB43 mice, except for Upk1b.The mean raw reads count of UPK3a in H 2 O 2 -treated mice was lower than that of control mice.The mean raw reads count of Cldn8 in SB43 mice was higher than that in H 2 O 2 -treated mice.No obvious differences were observed for proteoglycan core proteins and tight junction proteins.There were no previous reports on IC/PBS for GAG-synthesizing enzymes, but differences were found for Hyal1 and Xylt2.
A prestigious work by Gonzalez et al, reported that SB505124 improved urinary dysfunction by inhibiting hyperexcitation of bladder afferent nerves in cyclophosphamide-cystitis model [5,6].In the present study, SB431542 was found to have the best therapeutic effect among TGF-β inhibitors for the cystitis, despite being at the same concentration as other TGF-β inhibitors.SB431542 and SB505124 inhibited ALK4, 5, and 7, respectively, while Repsox had specific inhibition of ALK5, therefore inhibition of ALK5 may contribute to urinary function.However, we assume that the greater efficacy of SB431542 than Repsox and SB505124 is due to differences in the degree of inhibition of ALK4,7 and CK1α, β, δ [42][43][44].
TGF-β inhibitors have potential applications in a wide range of diseases.Preclinical and clinical trials have shown their usefulness in treatment for fibrosis and tumors, especially by enlarging to existing cancer therapies such as radiation, chemotherapy and tumor vaccines.However, TGF-β inhibitors has been reported to have tumorigenic potential, such as reversible cutaneous keratoacanthoma/squamous cell carcinoma and hyperkeratosis [45].In the present study, the blood samples showed no adverse effects on liver and kidney functions (ALT level is significantly elevated in H 2 O 2 /SB431542-treated group compared to control and the H 2 O 2 / saline-treated group, but the change is within the normal range) (S5 Fig).Also, no obvious adverse events such as weight loss were observed in the cystitis mice (S6 Fig) .To avoid adverse effects of TGF-β inhibitors, it may be safe to pre-select the patient population based on surrogate markers of TGF-β involvement in the disease process and possible adverse contraindications before initiating therapy [7].Peripheral blood may provide a useful non-invasive marker, such as the ability to quantitatively screen a patient's response to TGF-β inhibition based on measurement of P-SMAD2 levels in peripheral blood mononuclear cells [46].In addition, when TGF-β inhibitors are actually administered to IC/BPS patients, it would be preferable to inject the TGF-β inhibitor locally into the bladder wall, like Botox injections for intractable overactive bladder.Alternatively, TGF-β inhibitor may be infused into the bladder, similar to intravesical infusion of DMSO to treat Hunner type interstitial cystitis, to avoid side effects of systemic administration, as a unique administration route specific to the bladder [47].
As shown in Fig 1A, H2 O 2 -treated mice showed a significant increase in urinary frequency and a significant decrease in one-time voiding volume compared with non-treated and salinetreated mice.In H 2 O 2 -treated mice, the bladder urothelium was thin and damaged, and edematous thickening was observed in the submucosa 3 hours and 1 day after the H 2 O 2 infusion, compared with saline-treated mice (Fig1B).The urinary dysfunction was recovered to almost normal by the 8 th day after the H 2 O 2 infusion, while edematous thickening of the submucosal layer was seen even on day 8 (Fig1B).In previous report, the significant elevation of IL-1β mRNA expression was observed on the 7th day after the H 2 O 2 infusion[3].In the H 2 O 2induced cystitis model, the significant elevation of IL-1β mRNA expression was observed on day 14 (S2 Fig).Also, to use TGF-β inhibitors for the H 2 O 2 -induced cystitis model, TGF-β1 and TGF-β2 mRNA expression were measured.TGF-β1 and TGF-β2 mRNA expression was significantly elevated day 2 after the H 2 O 2 infusion compared to saline-infused mice.TGF-β1 mRNA significantly decreased on day 4 compared to day 2 mice (S2 Fig).Next, we examined the effects of TGF-β inhibitors on H 2 O 2 -induced cystitis in mice.Three TGF-β inhibitors, Repsox, SB431542, and SB505124, or saline was injected to the cystitis mice 30 min and 4 days after the H 2 O 2 infusion as shown in Fig 2. As shown in Figs 3A and S3, the intraperitoneal administration of all three TGF-β inhibitors significantly improved the tidal voiding volume on day 7 (Fig 3A).Repsox and SB431542, but not SB505124, significantly reduced the urinary frequency (Fig 3B).As shown in Fig 3C, any significant difference was not seen among the total urine

Fig 1 .
Fig 1. Intravesical infusion of H 2 O 2 caused IC/PBS-like urinary disturbance and histological changes in mice.Mice were given an intravesical infusion of 1.5% H 2 O 2 or saline through the urethra as described in the Materials and Methods.Control mice were inserted with a catheter through the urethra.(a) One and 8 days later, mice were subjected to voiding behavior analysis.Means ± sd. of tidal voiding volume (left) and urinary frequency (right) are shown (N = 3 mice for each group).**P<0.01 vs. Control.##P<0.01 vs. Saline.(b) After indicated periods, bladder was excised and tissue sections were stained with HE.Representative images of low (top panels) and high (bottom panels) magnifications are shown.Scale bars show 500 μm (top panels) and 100 μm (bottom panels).https://doi.org/10.1371/journal.pone.0293983.g001

Fig 4 .
Fig 4. RNA-seq analyses suggest molecular mechanisms through which SB431542 alleviated cystitis.H 2 O 2 -treated mice received intraperitoneal injections of SB431542 or saline as in Fig 3. On day 10, RNA was extracted from the bladder and examined by RNA sequencing.(a) Data of principal component analysis are shown.Blue, red and yellow dots represent control, H 2 O 2 /saline-treated, and H 2 O 2 /SB431542-treated groups, respectively.(b) Scatter plots are shown.Green lines show changes in expression with p = 0.01.ρ:Spearman's rank correlation coefficient (control group vs. H 2 O 2 /SB431542-treated group: R2 = 0.932, H 2 O 2 /saline-treated group vs. H 2 O 2 /SB431542-treated group: R2 = 0.878, control H 2 O 2 /saline-treated group vs. H 2 O 2 /saline-treated group: R2 = 0.765).(c) Biological processes suggested by Gene Ontology (GO) analysis are shown.GO terms that were changed more than 2-fold were selected, and the top 10 GO terms with the lowest p-values are drawn.(d) Heat maps and hierarchical clustering analyses comparing control, H 2 O 2 /saline and H 2 O 2 /SB431542 groups are shown.https://doi.org/10.1371/journal.pone.0293983.g004

Fig 5 .
Fig 5. SB431542 treatment restored enhanced expression of several genes.Bladder was excised from control, H 2 O 2 / saline-treated and H 2 O 2 /SB431542-treated mice as in Fig 4. RNA was extracted from the bladder, and RT-qPCR validation of the indicated genes were performed.Shown are as means ± sd. of relative mRNA levels (n = 4 mice).*p<0.05 and **p<0.01 between the indicated groups.https://doi.org/10.1371/journal.pone.0293983.g005